This invention was made with the support of NIH grant number RO1-CA-45814-01. The Government has certain rights in the invention.
The human hematopoietic system is populated by cells of several different lineages. These "blood cells" may appear in bone marrow, the thymus, lymphatic tissue(s) and in peripheral blood. Within any specific lineage, there are a number of maturational stages. In most instances, the more immature developmental stages occur within bone marrow while the more mature and final stages of development occur in peripheral blood.
There are two major lineages: The myeloid lineage which matures into red blood cells, granulocytes, monocytes and megakaryocytes; and the lymphoid lineage which matures into B lymphocytes and T lymphocytes. Within each lineage and between each lineage, antigens are expressed differentially on the surface and in the cytoplasm of the cells in a given lineage. The expression of one or more antigens and/or the intensity of expression can be used to distinguish between maturational stages within a lineage and between lineages.
Assignment of cell to lineage and to a maturational stage within a cell lineage indicates lineage commitment. There are cells, however, which are uncommitted to any lineage (i.e., the "progenitor" cell) and which, therefore, retain the ability to differentiate into each lineage. These undifferentiated, pluripotent progenitor cells will hereinafter be referred to as the "stem cells."
Therefore, all of mammalian hematopoietic cells can, in theory, be derived from a single stem cell. The stem cell is able to self-renew, so as to maintain a continuous source of pluripotent cells. In addition, when subject to particular environments and/or factors, the stem cells may differentiate to yield dedicated progenitor cells, which in turn may serve as the ancestor cells to a limited number of blood cell types. These ancestor cells will go through a number of stages before ultimately yielding a mature cell.
The benefit of obtaining a pure population of stem cells is most readily recognized in the field of gene therapy. Briefly, gene therapy can be used to treat specific diseases caused by a defect in a particular gene. For example, sickle cell anemia is caused by a defect in a single gene. The red blood cells of sickle cell patients contain this defective gene which, in turn, codes for a defective form of the protein hemoglobin. The defective form results in the clinical condition of sickle cell anemia. Sickle cell anemia cannot be "cured" by conventional drug therapies because the underlying defect is in the gene which is included within every cell.
Gene therapy seeks to replace or repopulate the cells of the hematopoietic system with cells that do not contain the defective gene but instead contain a "normal" gene. Using conventional recombinant DNA techniques, a "normal" gene is isolated, placed into a viral vector, and the viral vector is transfected into a cell capable of expressing the product coded for by the gene. The cell then must be introduced into the patient. If the "normal" gene product is produced, the patient is "cured" of the condition. The difficulty is that the transformed cells must be capable of continual regeneration as well as growth and differentiation.
Kwok et al., PNAS USA, 83, 4552 (1986), successfully demonstrated that gene therapy was possible using progenitor cells in dogs. Kwok et al. incorporated certain genes into the equivalent of lineage committed cells by retroviral transfection using standard recombinant DNA techniques and transplanted the transfected cells into the dogs. They obtained expression of the gene product(s) in cells isolated from the dogs. While the cells used by Kwok et al. are capable of growth and differentiation, they are not capable of self-renewal. Thus, any "cure" would be temporary. Stem cells, however, provide a better choice of cells in which to transfect a vector containing a "normal" gene. Stem cells have the capability not only of differentiating into cells of every lineage but also of self-renewal, thus establishing an unlimited supply of such cells. Therefore, by transplanting a stem cell, cells of every type in the hematopoietic system containing the "normal" gene will be continuously provided.
However, stem cells are usually in a resting state, which lowers the efficiency of transfection. Also, one of the most effective methods for viral transfection of hemotopoietic cells known thus far is co-cultivation of the target cells with "high titer, virus-producing cell lines." These virus-producing cell lines are often derived from other species, such as the mouse. Infusion of a human patient with a transfected stem cell population contamination by virus-producing cell lines, especially those derived from another species, is objectionable.
Furthermore, substantial problems have been encountered in (a) identifying the antigenic markers unique to stem cells, (b) isolating homogenous populations comprising substantial numbers of non-lineage committed, pluripotent stem cells and (c) maintaining and, possibly, expanding populations of human stem cells.
Difficulties are also presented by the fact that the stem cell population constitutes only a small percentage of the total number of leukocytes in bone marrow. I. L. Weissman et al. have reported that murine bone marrow cells contain only about 0.02-0.1% pluripotent stem cells. This group reported that Thy-1.sup.1o Lin.sup.- Sca2-1.sup.- murine bone marrow cells are a "virtually pure population of primitive myeloerythroid stem cells." Only 20-30 of these cells were sufficient to rescue one-half of a group of lethally-irradiated mice. See, Stanford University (published European Patent Application No. 341,966), and G. J. Spangrude et al., Science, 241, 58 (1988).
However, at the present time it is not known which antigens are present on stem cells alone or are also present on more differentiated progenitors. As in mice, one marker which has been indicated as present on human stem cells, CD34, is also found on a significant number of lineage committed progenitors. Another antigen which has been reported to provide for some enrichment of progenitor activity is Class II HLA (particularly a conserved DR epitope recognized by a monoclonal antibody designated J1-43). However, these markers are also found on numerous lineage committed hematopoietic cells. The Thy-1 molecule is a highly conserved protein present in the brain and in the hematopoietic system of rat, mouse and man. These species differentially express this antigen and the true function of this molecule is unknown. However, the Thy-1 molecule has been identified on rat and mouse hematopoietic stem cells. This protein is also believed to be present on most human bone marrow cells, but may be absent on stem cells.
Recently, a number of research groups have reported the use of these and other markers to isolate populations of mammalian bone marrow cell populations which are enriched to a greater or lesser extent in pluripotent stem cells. For example, in U.S. Pat. No. 4,714,680, Civin describes a differentiation antigen which is recognized by the monoclonal antibody designated My-10. In normal (i.e., non-leukemic) individuals, this antigen is found on progenitor cells within the hematopoietic system. Accordingly, Civin has described a population of progenitor stem cells which express the antigen recognized by My-10 (i.e., express the CD34 antigen), and has described a method of using My-10 to isolate stem cells for bone marrow transplantation. My-10 has been deposited with the American Type Culture Collection (Rockville, Md.) as HB-8483. My-10 is commercially available from Becton Dickinson Immunocytometry Systems ("BDIS") as anti-HPCA 1. However, using an anti-CD34 monoclonal antibody alone is not sufficient to distinguish between "stem cells," as described by Civin, and the true pluripotent stem cell, since B cells (CD19.sup.+) and myeloid cells (CD33.sup.+) make up 80-90% of the CD34.sup.+ population.
More recently, Becton Dickinson and Company (published European Patent Application No. 455,482) claimed a "substantially pure population of human cells containing pluripotent stem cells that express the CD34 antigen but lack expression of the CD38 antigen and other lineage associated antigens." To isolate this population of human pluripotent stem cells, a combination of anti-CD34 and anti-CD38 monoclonal antibodies are used to select those human progenitor stem cells that are CD34.sup.+ and CD38.sup.-. One method for the preparation of such a population of progenitor stem cells is to stain the cells with immunofluorescently labelled monoclonal antibodies. The cells then may be sorted by conventional flow cytometry wherein those cells that are CD34.sup.+ and those cells that are CD38.sup.- are selected for. Upon sorting, a substantially pure population of stem cells is reported to result.
Tsukamoto et al. (U.S. Pat. No. 5,061,620) disclose a method for the negative selection of differentiated and "dedicated" cells from human bone marrow to yield a population comprising "human hematopoietic stem cells with fewer than 5% lineage committed cells." The stem cells are characterized as being "for the most part" CD34.sup.+, CD3.sup.-, CD7.sup.-, CD8.sup.-, CD10.sup.-, CD14.sup.-, CD15.sup.-, CD19.sup.-, CD20.sup.-, CD33.sup.-, Class II HLA.sup.+ and Thy-1.sup.+.
C. Verfaillie et al., J. Exp. Med., 172, 509 (1990) reported that a two-step purification of low density human bone marrow cells by negative immunomagnetic selection and positive dual-color fluorescence activated cell sorting (FACS) yielded a Lin.sup.- CD34.sup.+ HLA/DR.sup.- cell fraction that was 420-fold enriched in pluripotent stem cells capable of initiating long-term bone marrow cultures (LTBMC); over unmanipulated bone marrow mononucleocytes (BMMNC) obtained after Ficoll-Hypaque separation. This group reported that the combination of positive selection for small blast-like cells that are CD34 antigen positive but HLA-DR antigen negative, combined with a more extensive negative selection to deplete the population of CD2, CD19 and CD71, results in an about two- to three-fold greater enrichment in pluripotent stem cells over that previously reported.
The development of cell culture media and conditions that will maintain stem cells in vitro for the extended periods of time required for the procedures involved in gene therapy, identification of growth factors, thorough characterization of cell morphologies and the like, has presented a unique set of obstacles. To date, successful in vitro stem cell cultures have depended on the ability of the laboratory worker to mimic the conditions which are believed to be responsible for maintaining stem cells in vivo.
For example, hematopoiesis occurs within highly dense cellular niches within the bone marrow in the adult and in similar niches within the fetal yolk sac and liver. Within these niches, stem cell differentiation is regulated, in part, through interactions with local mesenchymal cells or stromal cells. Mammalian hematopoiesis has been studied in vitro through the use of various long-term marrow culture systems. T. M. Dexter et al., in J. Cell Physiol., 91, 335 (1977) described a murine system from which spleen colony-forming units (CFU-S) and granulocyte/macrophage colony forming units (CFU-GM) could be detected for several months, with erythroid and megakaryocytic precursors appearing for a more limited time. Maintenance of these cultures was dependent on the formation of an adherent stromal cell layer composed of endothelial cells, adipocytes, reticular cells, and macrophages. These methods were soon adapted for the study of human bone marrow. Human long-term culture systems were reported to generate assayable hematopoietic progenitor cells for 8 or 9 weeks, and, later, for up to 20 weeks (See, S. Gartner et al., PNAS USA, 77, 4756 (1980); F. T. Slovick et al., Exp. Hematol., 12, 327 (1984)). Such cultures were also reliant on the preestablishment of a stromal cell layer which must frequently be reinoculated with large, heterogeneous populations of marrow cells. Hematopoietic stem cells have been shown to home and adhere to this adherent cell multi-layer before generating and releasing more committed progenitor cells (M. Y. Gordon et al., J. Cell. Physiol., 130, 150 (1987)).
Stromal cells are believed to provide not only a physical matrix on which stem cells reside, but also to produce membrane-contact signals and/or hematopoietic growth factors necessary for stem cell proliferation and differentiation. This heterogeneous mixture of cells comprising the adherent cell layer presents an inherently complex system from which the isolation of discrete variables affecting stem cell growth has proven difficult. Furthermore, growth of stem cells on a stromal layer makes it difficult to recover the hematopoietic cells or their progeny efficiently.
Recently, J. Brandt et al., in J. Clin. Invest., 86, 932 (1990), reported the maintenance of hematopoiesis of CD34.sup.+, DR.sup.-, CD15.sup.-, CD71.sup.- human marrow cells in liquid culture for up to 8 weeks, when the culture was supplemented with 48-hourly additions of recombinant IL-1.alpha., IL-3, IL-6 or granulocyte/macrophage colony-stimulating factor (GM-CSF). The establishment of an adherent cell layer was not observed, but cultures containing no exogenous cytokines produced clonogenic cells for only one week. However, even with the optimal cytokine combinations evaluated by Brandt et al., the progenitor cell (blast) population declined throughout the lifetime of these cultures, so that it is not clear that stem cell survival or proliferation is supported by this methodology.
Therefore, a need exists for methods for the long-term in vitro culture of human stem cells.